Distinct types of neurons are generated at specific locations during development of the central nervous system. These neurons then connect precisely with their targets to form a functional neural network. The goal of this project is to understand the molecular mechanisms that control neuronal cell fate determination and the effects of neuronal identity in axonal projection and target selection. We propose to study how neuronal subtype identities are acquired along the rostrocaudal axis in the developing spinal cord, focusing on the control of Hox gene expression and the functions of Hox genes in determining motor neuron identity and their peripheral projection using both chick and mouse as model systems. We will first examine if the signaling molecules, namely FGF, RA, and GDF11, which were responsible for the induction of profiled Hox-c protein expression in our in vitro culture system also have similar functions in vivo by introducing these factors and/or modified forms of their receptors into chick embryonic spinal cord using in ovo eleetroporation and analyze the changes in Hox-c protein expression. We will then evaluate the role of Cdx genes in mediating the functions of these external signals to induce profiled Hox-c protein expression. We will examine the effects of FGF, RA, and GDF11 on Cdx gene expression using both in vitro explant culture and in ovo electroporation. We will also test the ability of different Cdx genes to alter Hox-c gene expression profiles, using in ovo electroporation in chick embryos. Finally, to evaluate the function of Hox-c cluster genes in determining the rostrocaudal identity and peripheral projection of spinal motor neurons, we propose to generate motor neuron specific loss-of function mutations of Hox-c genes in mouse, as well as mis-express Hox-c genes in motor neurons of chick embryos. We will then analyze potential changes in motor neuron identity and projection during embryonic development in chick and mouse as well as examine motor/behavioral phenotypes in adult mice. These studies should contribute toward our understanding of the molecular mechanisms that control the generation of neuronal subtype identity and projections during spinal cord development, thus, providing the ground work for finding potential treatments for spinal cord injury and neurodegenerative diseases.